Investigating the contributions of astrocyte gap junctions to ALS disease progression

 描述(由申请人提供)：了解为什么大多数肌萎缩侧索硬化症(ALS)患者的疾病进展会随着时间的推移发生在连续的解剖区域，这是设计可在诊断后使用的疾病修改疗法的基本限制之一。来自野生型嵌合表达(WT)和运动神经元周围突变型SOD1星形胶质细胞的嵌合小鼠的研究表明，星形胶质细胞在发病后的疾病传播中发挥了作用。然而，星形胶质细胞功能障碍并不局限于转基因ALS啮齿动物模型，重要的是，无论是在原位检测还是在体外使用人类细胞，星形胶质细胞功能障碍都是ALS患者中最一致的观察结果之一。星形胶质细胞在中枢神经系统(CNS)中通过缝隙连接(GJS)和围绕中央毛孔排列的6个连接蛋白亚基组成的半通道形成高度耦合的细胞间网络。星形胶质细胞中的连接蛋白具有重要的作用：稳态缓冲、星形胶质细胞网络的同步化、神经元的代谢支持和血管生理的调节。它们还可以传播钙波，调节突触事件，或者通过半脑沟释放神经胶质递质。Cx43是星形胶质细胞中主要的连接蛋白，在中枢神经系统中广泛表达。我们的初步数据显示，Cx43在ALS模型以及ALS患者的人脊髓和皮质中表达上调。然而，这种Cx43上调在ALS中的功能意义尚未被研究。由于Cx43在星形胶质细胞连接和细胞-细胞信号转导中起作用，我们推测在ALS中星形胶质细胞Cx43表达上调可能与神经元丢失和邻近解剖疾病在ALS中的扩散有关。这项建议将在体外使用啮齿动物来源的星形胶质细胞和重要的是来自ALS患者的人IPSC来源的星形胶质细胞来研究这些变化的功能意义。此外，还将在体外研究Cx43上调ALS星形胶质细胞对野生型运动神经元丢失的具体影响。最后，我们将在ALS动物模型中利用多种遗传策略以及Cx43的局部敲除来解决Cx43的减少是否可以限制疾病的传播。这些研究将着眼于将关于Cx43介导的星形细胞对疾病进展的影响转化为潜在的潜在药理学策略的潜力，并在ALS诊断后进行干预。

 DESCRIPTION (provided by applicant): Understanding why disease progression in the majority of patients with Amyotrophic Lateral Sclerosis (ALS) occurs in contiguous anatomic regions over time is one of the fundamental limitations to designing disease modifying therapies that can be utilized after a diagnosis. Studies from chimeric mice with mosaic expression of wildtype (WT) and mutant SOD1 astrocytes surrounding motor neurons suggest that astrocytes play a role in disease propagation after onset. However, astrocyte dysfunction is not an observation merely limited to transgenic ALS rodent models but, importantly, has been one of the most consistent observations in humans with ALS when examined in situ as well as using human cells in vitro. Astrocytes form a highly coupled intercellular network in the central nervous system (CNS) through gap junctions (GJs) and hemichannels composed of 6 connexin subunits arranged around a central pore. Connexins in astrocytes have key roles: homeostatic buffering, synchronization of astrocyte networks, metabolic support for neurons, and regulation of vascular physiology. They can also propagate Ca2+ waves and modulate synaptic events or release gliotransmitters through hemichannels. Cx43 is the predominant connexin in astrocytes and is expressed ubiquitously in the CNS. Our preliminary data show that Cx43 is upregulated in ALS models as well as in human spinal cord and cortex in patients with ALS. Yet the functional significance of this Cx43 upregulation has not been studied in ALS. Because Cx43 has a role in astrocyte connectivity and cell-cell signaling, we hypothesize that astrocyte Cx43 upregulation in ALS may contribute to neuronal loss as well as contiguous anatomical disease spread in ALS. This proposal will address the functional significance of these changes in vitro using rodent-derived astrocytes and, importantly, human iPSC-derived astrocytes from ALS patients. The specific influences of Cx43 upregulation in ALS astrocytes on wildtype motor neurons loss will also be investigated in vitro. Finally, we will utilize a variety of genetic strategies as well as focal knockdown of Cx43 in ALS animal models, to address whether reductions in Cx43 can limit disease spread. These studies will occur with an eye towards the potential for translating what is learned about Cx43-mediated astrocytic influences on disease progression and spread to the development of potential pharmacological strategies for intervention after an ALS diagnosis.